Changes in environmental exposures over decades may influence the genetic architecture of severe spermatogenic failure.

STUDY QUESTION: Do the genetic determinants of idiopathic severe spermatogenic failure (SPGF) differ between generations? SUMMARY ANSWER: Our data support that the genetic component of idiopathic SPGF is impacted by dynamic changes in environmental exposures over decades. WHAT IS KNOWN ALREADY: The idiopathic form of SPGF has a multifactorial etiology wherein an interaction between genetic, epigenetic, and environmental factors leads to the disease onset and progression. At the genetic level, genome-wide association studies (GWASs) allow the analysis of millions of genetic variants across the genome in a hypothesis-free manner, as a valuable tool for identifying susceptibility risk loci. However, little is known about the specific role of non-genetic factors and their influence on the genetic determinants in this type of conditions. STUDY DESIGN, SIZE, DURATION: Case-control genetic association analyses were performed including a total of 912 SPGF cases and 1360 unaffected controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants had European ancestry (Iberian and German). SPGF cases were diagnosed during the last decade either with idiopathic non-obstructive azoospermia (n = 547) or with idiopathic non-obstructive oligozoospermia (n = 365). Case-control genetic association analyses were performed by logistic regression models considering the generation as a covariate and by in silico functional characterization of the susceptibility genomic regions. MAIN RESULTS AND THE ROLE OF CHANCE: This analysis revealed 13 novel genetic association signals with SPGF, with eight of them being independent. The observed associations were mostly explained by the interaction between each lead variant and the age-group. Additionally, we established links between these loci and diverse non-genetic factors, such as toxic or dietary habits, respiratory disorders, and autoimmune diseases, which might potentially influence the genetic architecture of idiopathic SPGF. LARGE SCALE DATA: GWAS data are available from the authors upon reasonable request. LIMITATIONS, REASONS FOR CAUTION: Additional independent studies involving large cohorts in ethnically diverse populations are warranted to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Overall, this study proposes an innovative strategy to achieve a more precise understanding of conditions such as SPGF by considering the interactions between a variable exposome through different generations and genetic predisposition to complex diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the "Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)" (ref. PY20_00212, P20_00583), the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. PID2020-120157RB-I00 funded by MCIN/ AEI/10.13039/501100011033), and the 'Proyectos I+D+i del Programa Operativo FEDER 2020' (ref. B-CTS-584-UGR20). ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, is also partially supported by the Portuguese Foundation for Science and Technology (Projects: UIDB/00009/2020; UIDP/00009/2020). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Study of SARS-CoV-2 in semen from asymptomatic donors with the presence of virus in nasopharyngeal swabs.

RESEARCH QUESTION: Is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present in semen samples from asymptomatic donors who have positive virus results from nasopharyngeal swabs? DESIGN: Nasopharyngeal PCR was performed on 1943 sperm donors between January 2021 and March 2022. The result was positive for 140 donations, and the presence of SARS-CoV-2 could be studied in cryopreserved semen from 84 of these donors. This included 67 participants in whom the quality of fresh semen could be compared with the previous donation, the day of the PCR-positive nasopharyngeal sampling and the first subsequent donation. Semen donations were cryopreserved following total semen (n = 26) or ready-to-use (n = 58) protocols. The presence of SARS-CoV-2 in cryopreserved samples was determined by reverse transcription PCR. Semen quality (volume, concentration and progressive motility) was evaluated in accordance with World Health Organization 2010 recommendations. RESULTS: SARS-CoV-2 virus was not detected in any cryopreserved total semen or ready-to-use samples. No significant differences in semen volume, concentration or progressive motility were observed between the last previous donation, the day of the positive PCR nasopharyngeal sampling and the first subsequent donation. CONCLUSIONS: The lack of detection of SARS-CoV-2 in semen samples from asymptomatic individuals infected with SARS-CoV-2 supports the safety of assisted human reproduction treatments using this type of sample.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Critical appraisal of the Vienna consensus: performance indicators for assisted reproductive technology laboratories.

The Vienna consensus, based on the recommendations of an expert panel, has identified 19 performance indicators for assisted reproductive technology (ART) laboratories. Two levels of reference values are established for these performance indicators: competence and benchmark. For over 10 years, the Spanish embryology association (ASEBIR) has participated in the definition and design of ART performance indicators, seeking to establish specific guidelines for ART laboratories to enhance quality, safety and patient welfare. Four years ago, ASEBIR took part in an initiative by AENOR, the Spanish Association for Standardization and Certification, to develop a national standard in this field (UNE 17900:2013 System of quality management for assisted reproduction laboratories), extending the former requirements, based on ISO 9001, to include performance indicators. Considering the experience acquired, we discuss various aspects of the Vienna consensus and consider certain discrepancies in performance indicators between the consensus and UNE 179007:2013, and analyse the definitions, methodology and reference values used.

Reliability and agreement on embryo assessment: 5 years of an external quality control programme.

An external quality-control programme for morphology-based embryo quality assessment, incorporating a standardized embryo grading scheme, was evaluated over a period of 5 years to determine levels of inter-observer reliability and agreement between practising clinical embryologists at IVF centres and the opinions of a panel of experts. Following Guidelines for Reporting Reliability and Agreement Studies, the Gwet index and proportion of positive (Ppos) and negative agreement were calculated. For embryo morphology assessment, a substantial degree of reliability was measured between the centres and the panel of experts (Gwet index: 0.76; 95% CI 0.70 to 0.84). The agreement was higher for good- versus poor-quality embryos. When multinucleation or vacuoles were observed, low levels of reliability were obtained (Ppos: 0.56 and 0.43, respectively). In blastocysts, the characteristic that presented the largest discrepancy was that related to the inner cell mass. In decisions about the final disposition of the embryo, reliability between centre and the panel of experts was moderate (Gwet index: 0.51; 95% CI 0.41 to 0.60). In conclusion, the ability of clinical embryologists to evaluate the presence of multinucleation and vacuoles in the early cleavage embryo, and to determine the category of the inner cell mass in blastocysts, needs to be improved.

Obstetric and neo-natal outcomes of ICSI cycles using pentoxifylline to identify viable spermatozoa in patients with immotile spermatozoa.

Pentoxifylline (PF) represents an effective tool in stimulating motility and identifying viable spermatozoa in intracytoplasmic sperm injection (ICSI) patients presenting exclusively with immotile spermatozoa. However, its use is not universally accepted for its possible detrimental effects on oocytes, embryos or newborns. To evaluate whether PF use may affect obstetrical/neo-natal outcomes, 102 patients achieving a clinical pregnancy after a PF-ICSI in four IVF units in Spain and Italy were followed up after delivery. Neo-natal malformations were classified according to the World Health Organization International Classification of Diseases (ICD-10, range Q00-Q99). Malformation rate was compared with data published by other groups regarding children conceived by conventional IVF or ICSI reporting a 5.3% and 4.4% frequency of ICD-10 codes, respectively. Of 134 clinical pregnancies, 122 babies (82 singletons and 40 twins) were registered. Among singletons, the rates of low birthweight (≤2500 g) and preterm birth (<37 weeks) were 6.1% and12%, respectively. Regarding malformation rate per live births, 4/122 (3.3%, 95% confidence interval: 0.9-8.2%) babies with ICD-10 malformations were recorded. This is the first report on neo-natal outcomes deriving from PF-ICSI. Although based on a limited cohort, results do not suggest an increase of adverse outcomes, including malformation rates, following this procedure.

Maximum number of children per sperm donor based on false paternity rate.

PURPOSE: The aim of this study is to estimate the weight of each relevant factor in such unions of inadvertent consanguinity and to determine a "reasonable" limit for the number of children per donor, matching the probability of inadvertent consanguinity arising from the use of sperm donor in assisted reproduction with that of such a union arising from false paternity. METHODS: In this study, we applied to Spanish data a mathematical model of consanguineous unions, taking into account the following factors: maximum number of live births/donor, fertility rate, average number of births per donor in a pregnancy, donor success rate, matings per phenotype, number of newborns/year, and number of donors needed in the population/year and births by false paternity. RESULTS: In Spain, the number of inadvertent unions between descendants of the same donor in Spain has been estimated at 0.4/year (one every two and a half years), although this frequency decreases as the reference population increases. On the other hand, the frequency of unions between family members due to false paternity has been estimated at 6.1/year. Thus, only 6% of such unions are due to the use of donor sperm. CONCLUSION: A total of 25 children per sperm donor are needed to align the probability of inadvertant consanguinity arising from the use of assisted reproduction with that due to false paternity. Therefore, we consider this number to be the maximum "reasonable" number of children born per donor in Spain.

Evolving minimum standards in responsible international sperm donor offspring quota.

An international working group was established with the aim of making recommendations on the number of offspring for a sperm donor that should be allowable in cases of international use of his sperm. Considerations from genetic, psychosocial, operational and ethical points of view were debated. For these considerations, it was assumed that current developments in genetic testing and Internet possibilities mean that, now, all donors are potentially identifiable by their offspring, so no distinction was made between anonymous and non-anonymous donation. Genetic considerations did not lead to restrictive limits (indicating that up to 200 offspring or more per donor may be acceptable except in isolated social-minority situations). Psychosocial considerations on the other hand led to proposals of rather restrictive limits (10 families per donor or less). Operational and ethical considerations did not lead to more or less concrete limits per donor, but seemed to lie in-between those resulting from the aforementioned ways of viewing the issue. In the end, no unifying agreed figure could be reached; however the consensus was that the number should never exceed 100 families. The conclusions of the group are summarized in three recommendations.

Cumulative live birth rate after two single frozen embryo transfers (eSFET) versus a double frozen embryo transfer (DFET) with cleavage stage embryos: a retrospective cohort study.

PURPOSE: According to the latest ART report for Europe, about 13% of pregnancies after frozen embryo transfer are multiple. Our objective was to analyse the impact on the multiple pregnancy rate of two eSFET (elective single frozen embryo transfers) versus a DFET (double frozen embryo transfer) in women aged under 38 years, who had not achieved pregnancy in their fresh transfer and who had at least two vitrified embryos of A/B quality. METHODS: This study was conducted from January 2010 to June 2013 at a public hospital. The couples were divided into three groups. Group DFET: the first cryotransfer of two embryos (105 women); cSFET group: the only cryotransfer of a single vitrified embryo (60 women); eSFET group, individually vitrified embryos: 20 patients included in a clinical trial of single-embryo fresh and frozen transfer and 21 patients who chose to receive eSFET. RESULTS: The clinical pregnancy rate was 38.1% in the DET group and the cumulative clinical pregnancy rate was 43.3% in the eSFET group. There were no significant differences between the DFET and eSFET groups (30.0 vs 34.1%) in cumulative live birth delivery rate. The rate of multiple pregnancies varied significantly between the DFET and eSFET groups (32.5 vs 0%, p < 0.05). CONCLUSIONS: For good-prognosis women aged under 38 years, taking embryo quality as a criterion for inclusion, an eSFET policy can be applied, achieving acceptable cumulative clinical pregnancy and live birth rates and reducing multiple pregnancy rates.

Common Variation in the PIN1 Locus Increases the Genetic Risk to Suffer from Sertoli Cell-Only Syndrome.

We aimed to analyze the role of the common genetic variants located in the PIN1 locus, a relevant prolyl isomerase required to control the proliferation of spermatogonial stem cells and the integrity of the blood-testis barrier, in the genetic risk of developing male infertility due to a severe spermatogenic failure (SPGF). Genotyping was performed using TaqMan genotyping assays for three PIN1 taggers (rs2287839, rs2233678 and rs62105751). The study cohort included 715 males diagnosed with SPGF and classified as suffering from non-obstructive azoospermia (NOA, n = 505) or severe oligospermia (SO, n = 210), and 1058 controls from the Iberian Peninsula. The allelic frequency differences between cases and controls were analyzed by the means of logistic regression models. A subtype specific genetic association with the subset of NOA patients classified as suffering from the Sertoli cell-only (SCO) syndrome was observed with the minor alleles showing strong risk effects for this subset (ORaddrs2287839 = 1.85 (1.17-2.93), ORaddrs2233678 = 1.62 (1.11-2.36), ORaddrs62105751 = 1.43 (1.06-1.93)). The causal variants were predicted to affect the binding of key transcription factors and to produce an altered PIN1 gene expression and isoform balance. In conclusion, common non-coding single-nucleotide polymorphisms located in PIN1 increase the genetic risk to develop SCO.

Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia.

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF. Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants. Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33-02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis. Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait.

Immune and spermatogenesis-related loci are involved in the development of extreme patterns of male infertility.

We conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DRß1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition.

Effect and in silico characterization of genetic variants associated with severe spermatogenic disorders in a large Iberian cohort.

BACKGROUND: Severe spermatogenic failure (SpF) represents the most extreme manifestation of male infertility, as it decreases drastically the semen quality leading to either severe oligospermia (SO, <5 million spermatozoa/mL semen) or non-obstructive azoospermia (NOA, complete lack of spermatozoa in the ejaculate without obstructive causes). OBJECTIVES: The main objective of the present study is to analyze in the Iberian population the effect of 6 single-nucleotide polymorphisms (SNPs) previously associated with NOA in Han Chinese through genome-wide association studies (GWAS) and to establish their possible functional relevance in the development of specific SpF patterns. MATERIALS AND METHODS: We genotyped 674 Iberian infertile men (including 480 NOA and 194 SO patients) and 1058 matched unaffected controls for the GWAS-associated variants PRMT6-rs12097821, PEX10-rs2477686, CDC42BPA-rs3000811, IL17A-rs13206743, ABLIM1-rs7099208, and SOX5-rs10842262. Their association with SpF, SO, NOA, and different NOA phenotypes was evaluated by logistic regression models, and their functional relevance was defined by comprehensive interrogation of public resources. RESULTS: ABLIM1-rs7099208 was associated with SpF under both additive (OR = 0.86, p = 0.036) and dominant models (OR = 0.78, p = 0.026). The CDC42BPA-rs3000811 minor allele frequency was significantly increased in the subgroup of NOA patients showing maturation arrest (MA) of germ cells compared to the remaining NOA cases under the recessive model (OR = 4.45, p = 0.044). The PEX10-rs2477686 SNP was associated with a negative testicular sperm extraction (TESE) outcome under the additive model (OR = 1.32, p = 0.034). The analysis of functional annotations suggested that these variants affect the testis-specific expression of nearby genes and that lincRNA may play a role in SpF. CONCLUSIONS: Our data support the association of three previously reported NOA risk variants in Asians (ABLIM1-rs7099208, CDC42BPA-rs3000811, and PEX10-rs2477686) with different manifestations of SpF in Iberians of European descent, likely by influencing gene expression and lincRNA deregulation.

Genetic Landscape of Nonobstructive Azoospermia and New Perspectives for the Clinic.

Nonobstructive azoospermia (NOA) represents the most severe expression of male infertility, involving around 1% of the male population and 10% of infertile men. This condition is characterised by the inability of the testis to produce sperm cells, and it is considered to have an important genetic component. During the last two decades, different genetic anomalies, including microdeletions of the Y chromosome, karyotype defects, and missense mutations in genes involved in the reproductive function, have been described as the primary cause of NOA in many infertile men. However, these alterations only explain around 25% of azoospermic cases, with the remaining patients showing an idiopathic origin. Recent studies clearly suggest that the so-called idiopathic NOA has a complex aetiology with a polygenic inheritance, which may alter the spermatogenic process. Although we are far from a complete understanding of the molecular mechanisms underlying NOA, the use of the new technologies for genetic analysis has enabled a considerable increase in knowledge during the last years. In this review, we will provide a comprehensive and updated overview of the genetic basis of NOA, with a special focus on the possible application of the recent insights in clinical practice.

Carriers of cystic fibrosis among sperm donors: complete CFTR gene analysis versus CFTR genotyping.

OBJECTIVE: To determine the frequency of cystic fibrosis (CF) carriers among sperm donors in Spain studied through a complete analysis of the CFTR gene and to compare the results with those that would have been obtained by the 4 genotyping panels of the CFTR gene most commonly used as a carrier test in the context of assisted reproduction in our country. DESIGN: Descriptive observational study. SETTING: Private center. PATIENTS: Nine hundred thirty-five sperm donors, from January 2014 to June 2019. INTERVENTION: None. MAIN OUTCOME MEASURE: Presence of pathogenic variants in the CFTR gene. RESULTS: 17% of the donors were carriers of at least 1 pathogenic variant in CFTR, with 39 different pathogenic variants detected. Only 4 of these 39 variants (10.27%) would have been detected by the 4 genotyping tests considered, and 22 variants (56.41%) would not have been detected by any of the genotyping tests. The pathogenic variants of the CFTR gene included in the different genotyping tests analyzed vary widely, and <50% are common to all of them. CONCLUSIONS: Although the was not based in the general population, these results show that the use of genotyping tests is associated with a high reproductive risk, because the rate of detection of CF carriers was lower when these panels were applied, in comparison with the complete study of the CFTR gene. We recommend that complete sequencing of the CFTR gene by next-generation sequencing be performed as a screening method for CF in sperm donors.

Intronic variation of the SOHLH2 gene confers risk to male reproductive impairment.

OBJECTIVE: To evaluate whether SOHLH2 intronic variation contributes to the genetic predisposition to male infertility traits, including severe oligospermia (SO) and different nonobstructive azoospermia (NOA) clinical phenotypes. DESIGN: Genetic association study. SETTING: Not applicable. PATIENT(S): Five hundred five cases (455 infertile patients diagnosed with NOA and 50 with SO) and 1,050 healthy controls from Spain and Portugal. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genomic DNA extraction from peripheral blood mononuclear cells, genotyping of the SOHLH2 polymorphisms rs1328626 and rs6563386 using the TaqMan allelic discrimination technology, case-control association analyses using logistic regression models, and exploration of functional annotations in publicly available databases. RESULT(S): Evidence of association was observed for both rs6563386 with SO and rs1328626 with unsuccessful sperm retrieval after testicular sperm extraction (TESE-) in the context of NOA. A dominant effect of the minor alleles was suggested in both associations, either when the subset of patients with the manifestation were compared against the control group (rs6563386/SO: P=.021, odds ratio [OR] = 0.51; rs1328626/TESE-: P=.066, OR = 1.46) or against the group of patients without the manifestation (rs6563386/SO: P=.014, OR = 0.46; rs1328626/TESE-: P=.012, OR = 2.43). The haplotype tests suggested a combined effect of both polymorphisms. In silico analyses evidenced that this effect could be due to alteration of the isoform population. CONCLUSION(S): Our data suggest that intronic variation of SOHLH2 is associated with spermatogenic failure. The genetic effect is likely caused by different haplotypes of rs6563386 and rs1328626, which may predispose to SO or TESE- depending on the specific allelic combination.

Evaluation of Male Fertility-Associated Loci in a European Population of Patients with Severe Spermatogenic Impairment.

Infertility is a growing concern in developed societies. Two extreme phenotypes of male infertility are non-obstructive azoospermia (NOA) and severe oligospermia (SO), which are characterized by severe spermatogenic failure (SpF). We designed a genetic association study comprising 725 Iberian infertile men as a consequence of SpF and 1058 unaffected controls to evaluate whether five single-nucleotide polymorphisms (SNPs), previously associated with reduced fertility in Hutterites, are also involved in the genetic susceptibility to idiopathic SpF and specific clinical entities. A significant difference in the allele frequencies of USP8-rs7174015 was observed under the recessive model between the NOA group and both the control group (p = 0.0226, OR = 1.33) and the SO group (p = 0.0048, OR = 1.78). Other genetic associations for EPSTI1-rs12870438 and PSAT1-rs7867029 with SO and between TUSC1-rs10966811 and testicular sperm extraction (TESE) success in the context of NOA were observed. In silico analysis of functional annotations demonstrated cis-eQTL effects of such SNPs likely due to the modification of binding motif sites for relevant transcription factors of the spermatogenic process. The findings reported here shed light on the molecular mechanisms leading to severe phenotypes of idiopathic male infertility, and may help to better understand the contribution of the common genetic variation to the development of these conditions.

Defining poor and optimum performance in an IVF programme.

BACKGROUND: At present there is considerable interest in healthcare administration, among professionals and among the general public concerning the quality of programmes of assisted reproduction. There exist various methods for comparing and analysing the results of clinical activity, with graphical methods being the most commonly used for this purpose. As yet, there is no general consensus as to how the poor performance (PP) or optimum performance (OP) of assisted reproductive technologies should be defined. METHODS: Data from the IVF/ICSI register of the Spanish Fertility Society were used to compare and analyse different definitions of PP or OP. The primary variable best reflecting the quality of an IVF/ICSI programme was taken to be the percentage of singleton births per IVF/ICSI cycle initiated. Of the 75 infertility clinics that took part in the SEF-2003 survey, data on births were provided by 58. A total of 25 462 cycles were analysed. The following graphical classification methods were used: ranking of the proportion of singleton births per cycles started in each centre (league table), Shewhart control charts, funnel plots, best and worst-case scenarios and state of the art methods. RESULTS: The clinics classified as producing PP or OP varied considerably depending on the classification method used. Only three were rated as providing 'PP' or 'OP' by all methods, unanimously. Another four clinics were classified as 'poor' or 'optimum' by all the methods except one. CONCLUSIONS: On interpreting the results derived from IVF/ICSI centres, it is essential to take into account the characteristics of the method used for this purpose.

Coverage and current practice patterns regarding assisted reproduction techniques.

Access to assisted reproduction techniques (ARTs) is highly variable worldwide. The aim of this paper is to analyse the association between systems for ART coverage, patterns of clinical practice, and outcomes for these procedures. We performed a retrospective analysis of ART activity records in the USA and Europe, and concluded that the availability of ART services, and the activity in this respect, is higher in public than in private coverage systems. Effectiveness decreases with greater coverage, but this occurs in parallel with lower numbers of embryos transferred and with multiple birth rates. Healthcare policy on assisted reproduction should take into account the necessary balance between the effectiveness of the treatment provided and its associated risks, the latter mainly concerning multiple pregnancies.

Inter-laboratory agreement on embryo classification and clinical decision: Conventional morphological assessment vs. time lapse.

The aim of this study is to determine inter-laboratory variability on embryo assessment using time-lapse platform and conventional morphological assessment. This study compares the data obtained from a pilot study of external quality control (EQC) of time lapse, performed in 2014, with the classical EQC of the Spanish Society for the Study of Reproductive Biology (ASEBIR) performed in 2013 and 2014. In total, 24 laboratories (8 using EmbryoScope™, 15 using Primo Vision™ and one with both platforms) took part in the pilot study. The clinics that used EmbryoScope™ analysed 31 embryos and those using Primo Vision™ analysed 35. The classical EQC was implemented by 39 clinics, based on an analysis of 25 embryos per year. Both groups were required to evaluate various qualitative morphological variables (cell fragmentation, the presence of vacuoles, blastomere asymmetry and multinucleation), to classify the embryos in accordance with ASEBIR criteria and to stipulate the clinical decision taken. In the EQC time-lapse pilot study, the groups were asked to determine, as well as the above characteristics, the embryo development times, the number, opposition and size of pronuclei, the direct division of 1 into 3 cells and/or of 3 into 5 cells and false divisions. The degree of agreement was determined by calculating the intra-class correlation coefficients and the coefficient of variation for the quantitative variables and the Gwet index for the qualitative variables. For both EmbryoScope™ and Primo Vision™, two periods of greater inter-laboratory variability were observed in the times of embryo development events. One peak of variability was recorded among the laboratories addressing the first embryo events (extrusion of the second polar body and the appearance of pronuclei); the second peak took place between the times corresponding to the 8-cell and morula stages. In most of the qualitative variables analysed regarding embryo development, there was almost-perfect inter-laboratory agreement among conventional morphological assessment (CMA), EmbryoScope™ and Primo Vision™, except for false divisions, vacuoles and asymmetry (users of all methods) and multinucleation (users of Primo Vision™), where the degree of agreement was lower. The inter-laboratory agreement on embryo classification according to the ASEBIR criteria was moderate-substantial (Gwet 0.41-0.80) for the laboratories using CMA and EmbryoScope™, and fair-moderate (Gwet 0.21-0.60) for those using Primo Vision™. The inter-laboratory agreement for clinical decision was moderate (Gwet 0.41-0.60) on day 5 for CMA users and almost perfect (Gwet 0.81-1) for time-lapse users. In conclusion, time-lapse technology does not improve inter-laboratory agreement on embryo classification or the analysis of each morphological variable. Moreover, depending on the time-lapse platform used, inter-laboratory agreement may be lower than that obtained by CMA. However, inter-laboratory agreement on clinical decisions is improved with the use of time lapse, regardless of the platform used.